Synthesis, Bioactivity and Crystal Structure Analysis of 2-(Benzo[d]isothiazol-3-yloxy)-N-(3-cyano-1-(4- fluorophenyl)-1H-pyrazol-5-yl) Acetamide①

YU Peng LI Xi HU Jun XU Yan-Hua②

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Synthesis, Bioactivity and Crystal Structure Analysis of 2-(Benzo[d]isothiazol-3-yloxy)-N-(3-cyano-1-(4- fluorophenyl)-1H-pyrazol-5-yl) Acetamide①

YU Penga, cLI Xib, cHU JundXU Yan-Huab, c②

a(210009)b(210009)c(210009)d(210009)

A novel benzisothiazolin-3-one derivative, 2-(benzo[d]isothiazol-3-yloxy)-N-(3- cyano-1-(4-fluorophenyl)-1H-pyrazol-5-yl) acetamide (8), was synthesized from the initial compound benzo[d]isothiazol-3(2H)-one (BIT) 1 and 4-fluoroaniline 3. The structure of the target compound 8 was determined by elemental analyses, IR and1H NMR. The single crystals of intermediate compound 6 and the target compound 8 were obtained and determined by X-ray diffraction analysis. The preliminary biological activity was also evaluated and the results showed the target compound exhibited a good anti-microbial activity.

benzisothiazol-3(2H)-one, pyrazol, synthesis, bioactivity, crystal structure

1 INTRODUCTION

Among various biological heterocyclic scaffolds, the derivatives of aryl pyrazol exhibited diverse bio- logical activities, e.g. insecticidal[1-4], antimicro- bial[5], anti-inflammatory[6-7]and herbicidal[8]. It’sworthnotingthat isothiazolinone derivatives are nowadays among the most important antimicrobial agents in the market. 1,2-benzisothiazol-3(2H)-one for example is a typical antimicrobial agent against bacteria and fungi[9-12]. Meanwhile, they also exhibi- ted diverse biological properties such as enzyme inhibitory activities[13-15]and anti-aggregating[16-18].

In connection with our research interest directed toward the synthesis and biological activity of novel benzisothiazol-3(2H)-one and aryl pyrazol amide derivatives, we herein synthesize a new amide com- pound containing the aryl pyrazol and isothiazo- linone. The synthesis route, crystal structure and potential antimicrobial activities of the target compound were described. The results showed that the target compound exhibit a good spectrum of activity againstand. The synthesis route is shown in Scheme 1.

2 EXPERIMENTA

2. 1 Materials and measurements

Benzo[d]isothiazol-3(2H)-one (BIT) and 4-fluoro- aniline were of analytical reagent grade (Aladdin Industrial Inc.). Unless otherwise noted, the other reagents and solvents were used as received from commercial suppliers.

Melting points were recorded on an X-4 binocular microscope melting point apparatus.1H NMR spectra were recorded on an Avance Bruker-500 instrument and chemical shifts in ppm were reported with TMS as the internal standard. IR spectra in KBr were recorded by a Perkin-Elmer PE-683 infrared spectrometer.Elemental analyses were performed on an Elementer Vario EL III elementary analysis instrument. Crystal structure determination was carried out on an Enraf-Nonius CAD-4 diffracto- meter.

The target compound 8 was preliminarily tested in laboratory in vitro to evaluate their anti-microbialactivities using the viable cells plate count me- thod[19]. Plate cultures were prepared as described in the literature[10]: 3 g beef, 5 g peptone, 5 g sodium chloride and 18 g agar. Then the medium mixed with different concentrations of the test compounds was autoclaved at 121 ℃ and 20 psi for 15 min to get toxic medium plane. After incubation at 37 ℃ for 24 h, colonies were counted.

2. 2 Synthesis and characterization

The target compound (8) was synthesized accor- ding to Scheme 1.

Scheme 1. Synthetic route of compound 8

A mixture of benzo[d]isothiazol-3(2H)-one (BIT, 1, 1.51 g, 0.01 mol) and sodiumhydride (0.29 g, 0.012 mol) in THF (50 mL) was stirred and refluxed for 2 hours. After completion of the reaction, the remaining THF was evaporated under negative pressure. The crude product was washed with ethanol and dried to afford compound 2 as white solid, m. p.: > 300 ℃.

To a mixture of compound3 (5.55 g, 0.05 mol) and ethanol (30 mL) was added concentratedhydro- chloricacid (14 mL) dropwise within 30 min, then the mixture was stirred for another 30 min. The mixture was added dropwise to the solution of sodiumnitrite (3.6 mol/L, 15 mL) within 1 h at 0 ℃ to obtain the crude compound 4as yellow oily liquid.

A dried 250 mL round-bottomed flask was added with ethyl 2,3-dicyanopropanoate (7.60 g, 0.05 mol), sodium acetate (12.30 g, 0.15 mol) and ethanol (20 mL). Subsequently, compound 4 (7.93 g, 0.05 mol) was added tothe mixture within 20 min and stirred by 2 h at 10 ℃. Then the mixture was extracted by dichloromethane (3’40 mL). The combined organic phase was added ammonia to adjust the pH to 9～10. After addition, the mixture was stirred at room temperature for 3 h. The organic phase was washed and concentrated. After cooling, the crude product was precipitated, filtered, washed with ethanol, dried and recrystallized from toluene to afford compound 6 as yellow solid.

5-Amino-1-(4-fluorophenyl)-1H-pyrazole-3-carbo-nitrile (6) with the yield of 80%. m. p.:116～118 ℃.1H NMR(DMSO-d6, 500MHz): 5.85 (s, 2H, NH2), 5.99 (s, 1H, CH), 7.61 (t, 4H, Ar-H); IR (KBr): 3135(N–H), 2920(C–H), 2243(C≡N), 1620(C=N), 1556(C=C), 1283(C–N), 1221(C-F) cm-1. Anal. Calcd. (%) for C10H7FN4: C, 59.40; H, 3.49; N, 27.71. Found (%): C, 59.38; H, 3.52; N, 27.68.

1.69 g (0.015 mol) of chloroacetylchloride was added dropwise in 30 mL dichloromethane solution of compound 6 (2.02 g, 0.01 mol) at 0～5 ℃, and the mixture thus obtained was stirred at room temperature for 2 h with triethylamine (1.52 g, 0.015 mol) as an acid acceptor. After completion of the reaction, the remaining THF was evaporated under reduced pressure to obtain the crude compound 7 as white solid. m. p.: 130～132 ℃.

Compounds 2 (2.09 g, 0.012 mol) and 7 (2.78 g, 0.01 mol) were dissolved in DMF (50 mL). The resulting mixture was refluxed for another 3.5 h with potassium iodide as catalyst. The reaction mixture was poured into water and stirred. Then the pre- cipitate formed was filtered off, washed with water, dried under reduced pressure and recrystallized from ethanol to get the target compound 8.

2-(Benzo[d]isothiazol-3-yloxy)-N-(3-cyano-1-(4-fluorophenyl)-1H-pyrazol-5-yl) acetamide (8) with the yield of 78%, m.p.: 176～178 ℃;1H NMR (CDCl3, 500 MHz): 5.16 (s, 2H, CH2), 7.18 (s, 1H, C–H), 6.93～7.85 (m, 8H, Ar–H), 8.54 (s, 1H, N–H); IR (KBr): 3202(N–H), 3058(C–H), 2242(C≡N), 1689, 1657(C=O), 1594(C=N), 1560(C=C), 1294(C–N), 1225(C–F), 650(C–S) cm-1. Anal. Calcd. (%) for C19H12FN5O2S: C, 58.01; H, 3.07; N, 17.80. Found (%): C, 58.08; H, 3.06; N, 17.71.

2. 3 X-ray structure determination

Colorless single crystals of 6 and 8 suitable for data collection were selected and mounted on a Bruker SMART APEX CCD diffractometer equip- ped with a graphite-monochromatic Mo-radia- tion (= 0.71073 ?) by using an-2scan mode at 293(2) K. Details of crystal data, data collection and refinement are given in Table 1. The calculations were performed with SHELXL-97 program[20]and expanded by using Fourier technique. The structure was refined by full-matrix least-squares techniques on2with anisotropic thermal parameters for all non-hydrogen atoms. The hydrogen atoms were added according to theoretical models.

Table 1. Crystal Data and Refinement Parameters for Compounds 6 and 8

3 RESULTS AND DISCUSSION

3. 1 Structure description of the compounds

Compound 6:The chemical diagram, molecular structure and packing diagram of 6 are shown in Scheme 1, Fig. 1 and Fig. 2, respectively. of 6 is shown in. The selected bond lengths and bond angles are given in Table 2. The details of hydrogen bonds are shown in Table 3. In the structure of 6, the C–C bond lengths range from 1.361(4) to 1.425(3) ?, almost equal to the values of typical bonds of aromatic structure[21]. The C(2)–N(3) bond length is 1.335(3) ?, between the C–N single bond (1.471 ?) and double bond (1.273 ?)[22], indicating the conjugation system of the five-membered ring. The C(4)–N(4) (1.369(3) ?) and C(4)–N(2) (1.371(3) ?) were both significantly shorter than C(2)–N(5) (1.426 (3) ?) due to the hybridization of two different types of C-atoms. The bond angles of benzene ring (C(5)/C(6)/C(7)/C(8)/C(9)/C(10)) varied from 117.7(2) to 123.4(2)o with the average of 120o.

Fig. 1. Molecular structure of 6

Fig. 2. Packing diagram of 6

Table 2. Selected Bond Lengths (?) and Bond Angles (°) for 6

Table 3. Hydrogen Bond Distances (?) and Bond Angles (o) for 6

Symmetry codes: (i) ?,+1/2, ?+1/2; (ii) ?+1, ?+1, ?; (iii) ?+1,+1/2, ?+1/2

In the crystal of compound 6, the benzene ring is, of course, planar, with an r.m.s deviation of 0.0110(3) ? and the pyrazol ring is a planar five-membered ring with an r.m.s deviation of 0.0032(2) ?. The dihedral angle between them is 38.8o. The intermolecular N(4)–H(4A)···N1i(d(D···A) 3.232(3) ?, ∠DHA = 158o), N(4)–H(4B)···Fii(d(D···A) = 3.131(3) ?, ∠DHA = 136o) and C(6)–H(6A)···Fiii(d(D···A) 3.410(3) ?, ∠DHA = 160o) hydrogen bonds (symmetry codes: (i) ?,+1/2, ?+1/2; (ii) ?+1, ?+1, ?; (iii) ?+1,+1/2, ?+1/2) expand the molecules into a 3D supramolecular architecture, and they may be effective in stabilizing the structure.

Compound 8:The chemical diagram, molecular structure and packing diagram of 8 are shown in Scheme 1, Fig. 3 and Fig. 4, respectively. The selected bond lengths and bond angles are given in Table 4. The details of hydrogen bonds are shown in Table 5. In the structure of compound 8, the bond lengths and bond angles within the heterocyclic system agree well with the values reported. The carbonyl C(11)–O(1) in 1.216(3) ? is found almost equal to a typical double bond (1.119 ?)[23]. However, C(13)–O(2) in 1.362(3) ? is a single carbon-oxygen bond. The C–N bond lengths are longer than those of 6. The bond angles of benzene rings A (C(1)/C(2)/C(3)/C(4)/C(5)/C(6)) and B (C(14)/C(15)/C(16)/C(17)/C(18)/C(19)) changed respectively from 119.95o to 120.26o and from 117.7(2)o to 123.4(2)o, both with the average of 120o.

In the crystal of compound 8, rings A (C(1)/ C(2)/C(3)/C(4)/C(5)/C(6)) and B (C(14)/C(15)/ C(16)/C(17)/C(18)/C(19)) are two planar benzene rings with r.m.s deviations of 0.0092(4) and 0.0024(2) ?, respectively. Rings C (C(7)/C(8)/ C(9)/N(2)/N(1)) and D (C(13)/C(14)/C(19)/S/N(5)) are planar five-member- ed pyrazol and thiazole rings, respectively. In ring C, the mean deviation from plane is 0.0035(2) ? and the standard deviations for the distances of C(7), C(8), C(9), N(2) and N(1) to the mean plane are 0.0009, 0.0024, –0.0049, 0.0053 and –0.0038 ?, correspondingly. In ring D, the mean deviation from the plane is 0.0017(3) ? and the C(13), C(14), C(19), S and N(5) atoms deviate from the mean plane by 0.0027, –0.0026, 0.0014, –0.0001 and –0.0016 ?, respectively. The dihedral angles between them are A/B = 61.6o, B/D = 0.2o, A/C = 56.6o and C/D = 17.5o. So, rings B and D are nearly coplanar. The intermolecular C–H···Oiand C–H···Niihydrogen bonds (symmetry codes: (i) ?, ??1, ?+1; (ii)+1,+1,+1) link the molecules to form a three-dimen- sional network (Fig. 4). Furthermore, the distance between the donor and acceptor of C(12)– H(12A)···O(1) is 3.375(4) ?, and C(18)– H(18A)···N(3) is 3.346(4) ?, simultaneously.

Table 4. Selected Bond Lengths (?) and Bond Angles (°) for 8

Table 5. Hydrogen Bond Distances (?) and Bond Angles (o) for 8

Symmetry codes: (i) ?, ??1, ?+1; (ii)+1,+1,+1

Fig. 3. Molecular structure of 8

Fig. 4. Packing diagram of 8

3. 2 Biological assay

Each antimicrobial experiment was conducted in six parallel samples. The values of antimicrobial activities are listed in Table 6. For the target com- pound was prepared as marine antifouling agent, the organisms used in the present investigation includedwhich was one of the typical gram positive bacteria existing in ocean sediments andwhich was one of the important parameters reflecting the seawater quality[10-12]. The results show that the target compounds have good antimicrobial activities against the tested organisms. When the solution concentration is below 100 ppm, the antimicrobial activities drop gradually, but are still better than the positive control (benzo[d]iso- thiazol-3(2H)-one).

Table 6. Antimicrobial Activities of the Target Compound (Inhibition %)

4 CONCLUSION

In conclusion, we have demonstrated the molecu- lar synthesis and antimicrobial activities of 2-(ben- zo[d]isothiazol-3-yloxy)-N-(3-cyano-1-(4-fluoro-phenyl)-1H-pyrazol-5-yl) acetamide 8. The preli- minary bioassay shows the target compound exhibits a favorable anti-microbial activity againstand. Expecting to find a new type of compounds containing aryl py- razol and isothiazolinone with high activities and low toxicities, further structural optimization and biological studies are well under way.

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20 February 2014;

25 March 2014 (CCDC 972550 for 6 and 972549 for 8)

① The generous financial support of this work from 2011 Key projects of Natural Science of Jiangsu province-owned colleges (No. 11KJA610001), Innovation project designated for graduate students of Jiangsu province (No. CXZZ13_0452), and the Postdoctoral research funding plan of Jiangsu province (No. 1202106C)

. Born in 1963, professor. E-mail: yhxu2008@163.com